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1、研究用肿瘤细胞系的质量控制和要求,中国医学科学院基础医学研究所刘玉琴,Some Facts about the use of cultured cells-Nobel prize,2001，Cell cycle2002,apoptosis2006,RNA Interference2007,homologous recombination and gene targeting2008,HPVcervical ca,HIV-aids2009,telomere&telomerase,Nobel prize winner,use in vitro cultured cells for their re
2、searches.,现代生命科学研究中培养细胞的使用越来越广泛,19692004：cell use increasing steadilyPapers use cells increase 22.5X biology,pharmacology and medicine-tumor research不可少的研究材料、对象、工具Use of contaminated or misidentified cells increase 10X,Common problems,microbiological contamination with particular respect to mycoplas
3、ma infections(incidence of ca.25%)cross-contamination by other cell lines and misidentification or misclassification(incidence of more than 15%)acquisition of genetic and epigenetic variations due to over-passaging of cell lines leading to a genetic drift of cell cultures and to the selection of alt
4、ered subclones.,Where from?,faulty cell culture techniques:shared reagents,repeated use of the same pipette during re-feeding operations and manipulation of multiple cultures at the same time without adequate isolation of one cell type from another.the introduction of false,contaminated or over-pass
5、aged cell lines from other sources into the cell culture laboratory(35%)Mislabeling,lack of awarenessIntentional co-cultivation during propagation of human stem or primary cells using a feeder layer derived from another species,细胞交叉污染历史回顾,1968,Apparent HeLa cell contamination of human heteroploid ce
6、ll lines.Nature.217:750-7511974，HeLa cess contaminating cultures around the world,Sciences 184:10591981,Contaminated cell cultures.Nature 289:2271981,cross-contamination of cells in culture.Sciences.212(4493):226-52,细胞交叉污染历史回顾,1999,False human hematopoietic cell lins:cross-cantaminations and misint
7、erpretations.Leukemia.13(10):1601-72003,False leukmia lymphoma cell lines:an update on over 500 cell lines.Leukemia 17,416-4262004,cell line cross-comtamination:how aware arein vitro cell dev biol anim.40(7):211-52007,Cases of mistaken identity.Sciences 315:928-9312009,Nature calls for global databa
8、se of cell line STR profiles,保藏机构公布的错误细胞1,保藏机构公布的错误细胞2,保藏机构公布的错误细胞3,保藏机构公布的错误细胞4,保藏机构公布的错误细胞5,保藏机构公布的错误细胞6,DSMZ 德国细胞库,550 leukemia and lymphoma cell lines submitted59/395(15%)by originators23/155(15%)by secondary sources,交叉污染细胞使用的后果,Erroneous results:Wasted research moneyPotential delay in discovery
9、,错误细胞使用,2000-2004：19/Int 407;45/WISH;59/Chang liver;470/Hep-2;556/KB;2004(Buehring),pubmed search 220publications involved,32%use HeLa cells,33%tested for authenticity,35%get cells from other labs.NCI panel of 60 cell lines were used for anticancer drug evaluation.Among them ADR-RES is acutally OVCA
10、R-8.It had been used in about 300 papers.,HeLa cervical cancer cells,Int-407(described as“non-transformed intestinal epithelial cells”)in Br.J.Cancer 101,1596(2009),EMBO J.22,5003(2003)and J.Biol.Chem.280,13538(2005)WISH(described as“non-transformed amniotic epithelial cells”)in Mol.Pharmacol.69,796
11、(2006),Endocrinology 147,2490(2006)and J.Biol.Chem.278,31731(2003)Chang liver(described as“normal liver cells”)in Oncogene 28,3526(2009),Proteomics 14,2885(2008)and J.Biol.Chem.279,28106(2000)HEp-2(described as“laryngeal cancer”)in Investig.New Drugs 26,111118(2008),Carcinogenesis 29,1519(2008)and J
12、.Biol.Chem.283,36272(2008)KB(described as“oral cancer”)in Biochem.Pharmacol.73,19011909(2007),Clin.Cancer Res.14,8161(2008)and J.Biol.Chem.280,23829(2005)HeLa,Int-407 and HEp-2 cells were used as three distinct cell lines in the same study in CancerRes.69,632(2009),Ecv-304,“人脐静脉内皮细胞”,日本实验室建立，ATCC、DS
13、MZ等机构有保藏，在19992000年，经STR检测，证明为T24(膀胱癌)，各自网站有公布。在实验室间广泛传播，2006y,全世界600余论文，利用该细胞进行研究，2/3在中国。Why?Donot know?No other suitable cellular modelExpensieve,can not afford,have to compromise,Ecv-304/T24,对细胞质量的要求,2007.10,Goslar,Germany，11ICCCScientist from ATCC proposed:(1)funding agencies,gov.or private requ
14、ire cell line authentication as a condition for the award of grant and contracts funds.(2)key scientific J as a condition for publication.(3)conference,workshop and/or training.(4)lab heads be encouraged to ensure QC measures.Authentic,Well characterizedThe problem of Sub-culturing,NIH NOTICE,2007.1
15、2,Require cell anthenticationFor grantFor paper,杂志及细胞使用者的要求Examples 1,Thp-1,2009-1-5cell line authentication,characterization and freedom from contamination This is an important issue,to which authors should pay particular attention.It is the authors responsibility to ensure that cell lines used in
16、any study(and the majority do involve them)are certified as being the designated type,that they have been checked to ensure they are free of contamination(e.g.mycoplasma),and that they have been used from young stock.These are the three criteria upon which good practice is based,as set out in the UK
17、CCC Guidelines on the use of cell lines(http:/).Attention should be paid to this in the Material and Methods section,and authors omitting to include evidence of good practice will find that their articles may be delayed until such guarantees are given.,Example 2,Reporting on Cell Line Use in AACR Jo
18、urnals,NEW!EFFECTIVE JULY 1,2009AACR strongly encourages the authentication of cell lines used in the research reported in its journals.If cell lines were used in the research,a statement addressing the following points must be included in the Materials and Methods section:1.From where and when the
19、cells were obtained 2.Whether the cell lines have been tested and authenticated 3.The method by which the cells were tested 4.How and when the cells were last tested,AACR Journals,Cancer Research Clinical Cancer Research Cancer Epidemiology,Biomarkers&Prevention，CEBP Cancer Prevention Research Molec
20、ular Cancer Research Molecular Cancer Therapeutics Cancer Reviews Online Prevention Portal,Example 2shelf life,If cells were obtained directly from a cell bank that performs cell line characterizations and passaged in the users laboratory for fewer than 6months after receipt or resuscitation,re-auth
21、orization is not required.In these cases,please include the method of characterization used by the cell bank.If cells were obtained directly from a cell bank that performs cell line characterizations and passaged in the users laboratory for fewer than 6months after receipt or resuscitation,re-author
22、ization is not required.In these cases,please include the method of characterization used by the cell bank.,Key milestones in the effort to address cell line misidentification,Hela cell line establishedfrom Henrietta Lacks,ATCC,American Type Culture Collection.NIH National Institutes of Health.STR,S
23、hort Tandem Repeat,Interspecies cross-Contaminationshown,ATCC starts Curating cell lines,Stan Gartler shows intraspeciesCross-contamination betweenHuman cell lines,Nelson-Ross confirms&estends Stan Gartlers findings,John Maddox describesIndividual like Nelson-RossAs self-appointed vigilantes,1981-20
24、05 crossContamination spreeds Beyongd HeLa cells,NIH terminatescontract of Nelson-Ross,NIH issues guidelinesTo avoid the use of misidentified cell lines,Roland Nardone starts the Second crusade agsinst Corss contamination,Nature calls for aGlobal database ofCell lines STR profies,1952 1958 1962 1966
25、 1974 1980 1981 2005 2007 2009,Hela cell line establishedfrom Henrietta Lacks,ATCC starts Curating cell lines,Hela cell line establishedfrom Henrietta Lacks,ATCC starts Curating cell lines,Hela cell line establishedfrom Henrietta Lacks,ATCC starts Curating cell lines,Nelson-Ross confirms&estends Sta
26、n Gartlers findings,Hela cell line establishedfrom Henrietta Lacks,ATCC starts Curating cell lines,Nelson-Ross confirms&estends Stan Gartlers findings,Hela cell line establishedfrom Henrietta Lacks,ATCC starts Curating cell lines,Nelson-Ross confirms&estends Stan Gartlers findings,Hela cell line est
27、ablishedfrom Henrietta Lacks,ATCC starts Curating cell lines,1981-2005 crossContamination spreeds Beyongd HeLa cells,Nelson-Ross confirms&estends Stan Gartlers findings,Hela cell line establishedfrom Henrietta Lacks,ATCC starts Curating cell lines,NIH issues guidelinesTo avoid the use of misidentifi
28、ed cell lines,1981-2005 crossContamination spreeds Beyongd HeLa cells,Nelson-Ross confirms&estends Stan Gartlers findings,Hela cell line establishedfrom Henrietta Lacks,ATCC starts Curating cell lines,1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,Interspecies cross-Contaminationshown,1952 1958 1
29、962 1966 1974 1980 1981 2005 2007 2009,Stan Gartler shows intraspeciesCross-contamination betweenHuman cell lines,Interspecies cross-Contaminationshown,1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,Stan Gartler shows intraspeciesCross-contamination betweenHuman cell lines,Interspecies cross-Cont
30、aminationshown,1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,John Maddox describesIndividual like Nelson-RossAs self-appointed vigilantes,Stan Gartler shows intraspeciesCross-contamination betweenHuman cell lines,Interspecies cross-Contaminationshown,1952 1958 1962 1966 1974 1980 1981 2005 2007
31、2009,John Maddox describesIndividual like Nelson-RossAs self-appointed vigilantes,Stan Gartler shows intraspeciesCross-contamination betweenHuman cell lines,Interspecies cross-Contaminationshown,1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,NIH terminatescontract of Nelson-Ross,John Maddox descr
32、ibesIndividual like Nelson-RossAs self-appointed vigilantes,Stan Gartler shows intraspeciesCross-contamination betweenHuman cell lines,Interspecies cross-Contaminationshown,1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,NIH terminatescontract of Nelson-Ross,John Maddox describesIndividual like Ne
33、lson-RossAs self-appointed vigilantes,Stan Gartler shows intraspeciesCross-contamination betweenHuman cell lines,Interspecies cross-Contaminationshown,1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,Roland Nardone starts the Second crusade agsinst Corss contamination,NIH terminatescontract of Nels
34、on-Ross,John Maddox describesIndividual like Nelson-RossAs self-appointed vigilantes,Stan Gartler shows intraspeciesCross-contamination betweenHuman cell lines,Interspecies cross-Contaminationshown,1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,Roland Nardone starts the Second crusade agsinst Cor
35、ss contamination,NIH terminatescontract of Nelson-Ross,John Maddox describesIndividual like Nelson-RossAs self-appointed vigilantes,Stan Gartler shows intraspeciesCross-contamination betweenHuman cell lines,Interspecies cross-Contaminationshown,1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,Natur
36、e calls for aGlobal database ofCell lines STR profies,Roland Nardone starts the Second crusade agsinst Corss contamination,NIH terminatescontract of Nelson-Ross,John Maddox describesIndividual like Nelson-RossAs self-appointed vigilantes,Stan Gartler shows intraspeciesCross-contamination betweenHuma
37、n cell lines,Interspecies cross-Contaminationshown,1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,Requirements for cell authentication,Morphology check by microscopeGrowth curve analysis Detection of mycoplasmaSpecies verification by IsoenzymologyIdentity verification with STR analysisSpecific pr
38、operties conformation,Detection of cross-comtamination,STR PROFILING METHODOLOGY,ADVANTAGES,How to avoid cross-contaminations,Cell cultures received from sources other than recognized cell banks should generally be tested for identity,contaminations,and suitability of the cells for the assays planed
39、.The mycoplasma and cross-contaminations can also occur during cultivation and therefore the cell culture quality tests should be performed at regular intervals.Simultaneous handling of different cultures under the laminar flow hood by one or more persons the use of a single flask of medium for diff
40、erent cell lines.The prophylactic use of antibiotic Avoid the negligent and erroneous labelling of cell culture vessels and cryo-ampoules.,Resources for Authors,UKCCCR Guidelines for the Use of Cell Lines in Cancer Research,British J of Cancer(2000)82(9);14951509.Cell Line Verification Test Recommen
41、dations,ATCC Technical Bulletin No.8(2008).Cell Line Authentication Methods Cabrera CM et al.,Identity Test:Determination of Cell Line Cross-Contamination,Cytotechnology(2006)51:4550.Masters JR et al.,Short Tandem Repeat Profiling Provides an International Reference Standard for Cell Lines,PNAS(2001)98(14):801217.http:/,谢谢！,
